1 Overview

# Read in  a tx2gene file with transcript identifiers in the first column and gene identifiers in the second column
tx2gene <- hsdb[, c("tx_id", "gene_id")]


# Run tximport
txi_file <- "data/txi.day8.RDS"
if (!rebuild_rds & file.exists(txi_file)){
    txi <- readRDS(txi_file)
}else{
    files <- files[rownames(meta)]
    txi <- tximport(files, 
                type = "salmon", 
                tx2gene = tx2gene, 
                countsFromAbundance = "lengthScaledTPM", 
                ignoreTxVersion = FALSE)
    saveRDS(txi, txi_file)
}

# Look at the counts
class(txi)
## [1] "list"
attributes(txi)
## $names
## [1] "abundance"           "counts"              "length"             
## [4] "countsFromAbundance"
txi$counts %>% View()

2 Checking to see that the transcript to gene mapping is correct

When you have annotations that are from a different source from your reference you can run into problems (i.e lose genes). Some checks you can do before proceeding:

  1. Look at the dimensions of your count matrix. Do you have ~20k genes present? dim(txi$counts)
  2. When running tximport() you will get a message in your console. If you see something like transcripts missing from tx2gene start troubleshooting.
dim(txi$counts)
## [1] 58735    22

3 Sanity check that metadata matches your expression

It is always a good idea to check if:

  1. Do you have expression data for all samples listed in your metadata?
  2. Are the samples in your expression data in the same order as your metadata?
### Check that sample names match in both files
all(colnames(txi$counts) %in% rownames(meta))
## [1] TRUE
### Check that sample names match in both files
all(colnames(txi$counts) %in% rownames(meta))
## [1] TRUE
### Check that all samples are in the same order
meta <- meta[colnames(txi$counts),]
all(colnames(txi$counts) == rownames(meta))
## [1] TRUE

4 Run DESeq2

estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

5 Wald test

Here we subset protein coding genes.

## Create DESeq2Dataset object
dds_file <- "data/dds.day8.RDS"
meta$treatment <- as.factor(meta$treatment)
meta$response <- as.factor(meta$response)
meta$er <- as.factor(meta$er)
meta$date_of <- as.factor(meta$date_of)
meta$tumor_percentage <- as.factor(meta$tumor_percentage)
meta$tumor_percentage_high <- as.factor(meta$tumor_percentage_high)
meta$tumor_percentage_high <- relevel(meta$tumor_percentage_high, "low")


if (remove_cases_2_19){
   non_responders <- meta %>% dplyr::filter(study_id %in% c(2, 19)) %>% row.names() 
}

if (!rebuild_rds & file.exists(dds_file)){
    dds <- readRDS(dds_file)
}else{
    dds <- DESeqDataSetFromTximport(txi,
                                colData = meta, 
                                design = ~response)
    
    if (remove_cases_2_19){
        dds <- dds[,!colnames(dds) %in% non_responders]
    }
    
    design(dds) <- formula(~response + er + tumor_percentage_high + date_of)
    
    # subset protein-coding genes
    pc_genes <- intersect(protein_coding_genes$ensembl_gene_id, row.names(dds))
    dds <- dds[pc_genes,]
    # 100 reads / 20 samples
    keep <- rowSums(counts(dds)) >= 100
    dds <- dds[keep,]

    # Run DESeq2
    dds <- DESeq(dds)
    saveRDS(dds, dds_file)
}

6 DEGreport QC

6.1 Size factor QC - samples 1-20

counts <- counts(dds, normalized = TRUE)
design <- as.data.frame(colData(dds))
degCheckFactors(counts[, 1:20])

7 Mean-Variance QC plots

7.1 response

res <- results(dds)
degQC(counts, design[["response"]], pvalue = res[["pvalue"]])

7.2 ER

degQC(counts, design[["er"]], pvalue = res[["pvalue"]])

7.3 tumor_percentage_high

degQC(counts, design[["tumor_percentage_high"]], pvalue = res[["pvalue"]])

8 Covariates effect on count data

mdata <- colData(dds) %>%  as.data.frame()  %>% 
  dplyr::select(response, er, date_of, tumor_percentage_high)

#resCov <- degCovariates(log2(counts(dds)+0.5), mdata)
mdata %>% ggplot(aes(tumor_percentage_high, fill = response)) + geom_bar(position = "dodge2")

9 Covariates correlation with metrics

cor <- degCorCov(mdata)

mdata %>% ggplot(aes(date_of, fill = response)) + geom_bar(position = "dodge2")

10 Sample-level QC analysis

### Transform counts for data visualization (unsupervised analysis)
rld_file <- "data/rld.day8.RDS"
if (!rebuild_rds & file.exists(rld_file)){
    rld <- readRDS(rld_file)
}else{
    rld <- rlog(dds, blind = TRUE)
    saveRDS(rld, rld_file)
}
class(rld) # what type of object is this
## [1] "DESeqTransform"
## attr(,"package")
## [1] "DESeq2"
# we also need just a matrix of transformed counts
rld_mat <- assay(rld)

10.1 PCA - response

# Use the DESeq2 function
plotPCA(rld, intgroup = c("response")) + geom_label_repel(aes(label = name))

10.2 PCA - ER

# Use the DESeq2 function
plotPCA(rld, intgroup = c("er")) + geom_label_repel(aes(label = name))

10.3 PCA - tumor_percentage

# Use the DESeq2 function
plotPCA(rld, intgroup = c("tumor_percentage")) + geom_label_repel(aes(label = name))

10.4 PCA - tumor_percentage_high

# Use the DESeq2 function
plotPCA(rld, intgroup = c("tumor_percentage_high")) + geom_label_repel(aes(label = name))

10.5 PCA - date_of

# Use the DESeq2 function
plotPCA(rld, intgroup = c("date_of")) + geom_label_repel(aes(label = name))

11 Inter-correlation analysis

11.1 Without study_id

# Correlation matrix
rld_cor <- cor(rld_mat)

meta$study_id <- as.factor(meta$study_id)
# Create annotation file for samples
annotation <- meta[, c("response", "er", "tumor_percentage_high", "date_of")]

# Change colors
heat.colors <- brewer.pal(6, "Blues")

# Plot heatmap
pheatmap(rld_cor, 
         annotation = annotation, 
         border = NA,
         fontsize = 20)

11.2 With study_id

# Correlation matrix
rld_cor <- cor(rld_mat)

meta$study_id <- as.factor(meta$study_id)
# Create annotation file for samples
annotation <- meta[, c("response", "er", "tumor_percentage_high", "date_of", "study_id")]

# Change colors
heat.colors <- brewer.pal(6, "Blues")

# Plot heatmap
pheatmap(rld_cor, 
         annotation = annotation, 
         border = NA,
         fontsize = 20)

12 Response pCR vs non-pCR for Day 8 - see Table13

13 ER : Positive vs Negative for Day8 - Table 14

14 tumor_percentage_high : High vs Low for Day8- Table 15

15 date_of: 20180323 vs 20180228 - for Day8: Table 16

16 Visualization

Gene example

d <- plotCounts(dds, 
                gene = "ENSG00000130234", 
                intgroup = "response", 
                returnData = TRUE)

ggplot(d, aes(x = response, y = count)) + 
     geom_point(position = position_jitter(w = 0.1, h = 0)) +
     geom_text_repel(aes(label = rownames(d))) + 
     theme_bw(base_size = 10) +
     ggtitle("ACE2") +
     theme(plot.title = element_text(hjust = 0.5)) +
     scale_y_log10()

# Add a column for significant genes
resResponse_tb_vis <- resResponse_tb %>% mutate(threshold = padj < 0.01)

# don't show labels for lfc < 1.5
resResponse_tb_vis$symbol <- ifelse((abs(resResponse_tb_vis$log2FoldChange) > 1.5),   
                                resResponse_tb_vis$symbol, "") 


# don't show labels for non-significant genes
resResponse_tb_vis$symbol <- ifelse(resResponse_tb_vis$threshold,   
                                    resResponse_tb_vis$symbol, "") 

#fig4b
# suggested by Jennifer
dont_show <- c("C20orf204", "B4GALNT4", "SMIM34B", "ARHGAP33", 
               "ATRNL1", "CROCC2", "CEL", "FLG", "KIAA1549L", 
               "AHRR", "SLC25A27", "CPLX1", "SNX10", "STX11",
               "FUCA1", "ACP5", "MS4A4A", "GM2A", "TYROBP", "GBP5", 
               "SASH3", "CTSL", "TFEC", "DNASE2", "IGSF6", "IFI30", "LGALS3",
               "FCER1G", "VNN2", "SELPLG", "GPX1", "TAGAP", "PRDX1", "SLAMF8",
               "SSR4", "LST1", "VMO1", "OMD", "PRB2", "TBX21", "LIPA", "COMMD3-BMI1",
               "SOWAHD", "LGALS2", "SLAMF6", "SH2D1A", "ALDH2", "PTPRCAP")

resResponse_tb_vis[resResponse_tb_vis$symbol %in% dont_show,]$symbol <- ""

# no highlighting
#resResponse_tb_vis$highlight <- ifelse(resResponse_tb_vis$symbol %in% genes_to_show, 
#                                    "yes", "no")

# a handful of extreme genes are not shown, like PRSS33
fig4b <- ggplot(resResponse_tb_vis,
       aes(log2FoldChange, -log10(padj), label = symbol)) +
  geom_point(aes(colour = threshold)) +
  ggtitle("Response pCR vs non-pCR") +
  xlab("log2 fold change") + 
  ylab("-log10 adjusted p-value") +
  scale_x_continuous(limits = c(-10, 10)) +
  scale_y_continuous(limits = c(0, 7)) +
  theme(legend.position = "none",
        plot.title = element_text(size = rel(1.5), hjust = 0.5),
        axis.title = element_text(size = rel(1.25)),
        panel.grid.major = element_blank(), 
        panel.grid.minor = element_blank(),
        panel.border = element_blank(),
        panel.background = element_blank()) +
  geom_label_repel(aes(label = symbol), 
                  data = subset(resResponse_tb_vis, log2FoldChange > 0),
                      size = 5, 
                      min.segment.length = 0,
                      segment.size = 0.1,
                      segment.color = "grey50",
                      direction     = "both",
                      max.overlaps = Inf,
                      segment.linetype = 5,
                      max.iter =  Inf,
                      hjust = "right",
                      xlim = c(3, 10),
                      force = 5,
                      force_pull = 0
                  ) +
  geom_label_repel(aes(label = symbol), 
                  data = subset(resResponse_tb_vis, log2FoldChange < 0),
                      size = 5, 
                      min.segment.length = 0,
                      segment.size = 0.1,
                      segment.color = "grey50",
                      direction     = "both",
                      max.overlaps = Inf,
                      segment.linetype = 5,
                      max.iter =  Inf,
                      hjust = "left",
                      xlim = c(-9, -4),
                      force = 5,
                      force_pull = 0
                  )

 # nudge_x = 9 - subset(resResponse_tb_vis, log2FoldChange > 0)$log2FoldChange,
#force_pull = 0, # attraction between label and point
 #                     force = 0.5 # repulsion between labels
#point.padding = 1,,
#                  point.size = NA,
#                  arrow = arrow(length = unit(0.015, "npc")),
#                  segment.linetype = 1,
#                  box.padding = 0.5,
#                  force_pull = 0,
#                  force = 1

saveRDS(fig4b, "data/fig4b.RDS")  
fig4b

# Add a column for significant genes
resER_tb <- resER_tb %>% mutate(threshold = padj < 0.01)

ggplot(resER_tb) +
  geom_point(aes(x = log2FoldChange, y = -log10(padj), colour = threshold)) +
  ggtitle("ER: Positive vs Negative") +
  xlab("log2 fold change") + 
  ylab("-log10 adjusted p-value") +
  scale_x_continuous(limits = c(-10,10)) +
  theme(legend.position = "none",
        plot.title = element_text(size = rel(1.5), hjust = 0.5),
        axis.title = element_text(size = rel(1.25)))

# Add a column for significant genes
resTP_tb <- resTP_tb %>% mutate(threshold = padj < 0.01)

ggplot(resTP_tb) +
  geom_point(aes(x = log2FoldChange, y = -log10(padj), colour = threshold)) +
  ggtitle("Tumor_percentage_high: High vs Low") +
  xlab("log2 fold change") + 
  ylab("-log10 adjusted p-value") +
  scale_x_continuous(limits = c(-10,10)) +
  theme(legend.position = "none",
        plot.title = element_text(size = rel(1.5), hjust = 0.5),
        axis.title = element_text(size = rel(1.25)))

# Add a column for significant genes
resDO_tb <- resDO_tb %>% mutate(threshold = padj < 0.01)

ggplot(resDO_tb) +
  geom_point(aes(x = log2FoldChange, y = -log10(padj), colour = threshold)) +
  ggtitle("Dafe of: 20180323 vs 20180228") +
  xlab("log2 fold change") + 
  ylab("-log10 adjusted p-value") +
  scale_x_continuous(limits = c(-10,10)) +
  theme(legend.position = "none",
        plot.title = element_text(size = rel(1.5), hjust = 0.5),
        axis.title = element_text(size = rel(1.25)))

17 Heatmaps

# Create a matrix of normalized expression
sig_up <- resResponse_tb_significant %>% arrange(-log2FoldChange) %>% head(50) %>% pull(gene)
sig_down <- resResponse_tb_significant %>% arrange(log2FoldChange) %>% head(50) %>% pull(gene)
sig <- c(sig_up, sig_down)

row_annotation <- gene_symbol %>% 
                    as_tibble() %>% 
                    dplyr::filter(gene_id %in% sig)

plotmat <- txi$abundance[c(sig_up, sig_down),] %>% as.data.frame() %>% 
          rownames_to_column(var = "ensembl_gene_id") %>% 
          left_join(gene_symbol, by = c("ensembl_gene_id" = "gene_id")) %>% 
          drop_na(symbol)

plotmat$ensembl_gene_id <- NULL

plotmat <- plotmat %>% column_to_rownames(var = "symbol") %>% as.matrix()

# Color palette
heat.colors <- brewer.pal(6, "YlOrRd")

# Plot heatmap
pheatmap(plotmat, 
         scale = "row", 
         show_rownames = TRUE,
         border = FALSE,
         annotation = meta[, c("response"), drop = FALSE],
         main = "Top 50 Up- and Down- regulated genes in Response: pCR vs non-pCR",
         fontsize = 20)

# Create a matrix of normalized expression
sig_up <- resER_tb_significant %>% arrange(-log2FoldChange) %>% head(50) %>% pull(gene)
sig_down <- resER_tb_significant %>% arrange(log2FoldChange) %>% head(50) %>% pull(gene)
sig <- c(sig_up, sig_down)

row_annotation <- gene_symbol %>% 
                    as_tibble() %>% 
                    dplyr::filter(gene_id %in% sig)

plotmat <- txi$abundance[c(sig_up, sig_down),] %>% as.data.frame() %>% 
          rownames_to_column(var = "ensembl_gene_id") %>% 
          left_join(gene_symbol, by = c("ensembl_gene_id" = "gene_id")) %>% 
          drop_na(symbol)

plotmat$ensembl_gene_id <- NULL

plotmat <- plotmat %>% column_to_rownames(var = "symbol") %>% as.matrix()

# Color palette
heat.colors <- brewer.pal(6, "YlOrRd")

# Plot heatmap
pheatmap(plotmat, 
         scale = "row", 
         show_rownames = TRUE,
         border = FALSE,
         annotation = meta[, c("er"), drop = FALSE],
         main = "Top 50 Up- and Down- regulated genes in ER: positive vs negative",
         fontsize = 20)

# Create a matrix of normalized expression
sig_up <- resTP_tb_significant %>% arrange(-log2FoldChange) %>% head(50) %>% pull(gene)
sig_down <- resTP_tb_significant %>% arrange(log2FoldChange) %>% head(50) %>% pull(gene)
sig <- c(sig_up, sig_down)

row_annotation <- gene_symbol %>% 
                    as_tibble() %>% 
                    dplyr::filter(gene_id %in% sig)

plotmat <- txi$abundance[c(sig_up, sig_down),] %>% as.data.frame() %>% 
          rownames_to_column(var = "ensembl_gene_id") %>% 
          left_join(gene_symbol, by = c("ensembl_gene_id" = "gene_id")) %>% 
          drop_na(symbol)

plotmat$ensembl_gene_id <- NULL

plotmat <- plotmat %>% column_to_rownames(var = "symbol") %>% as.matrix()

# Color palette
heat.colors <- brewer.pal(6, "YlOrRd")

# Plot heatmap
pheatmap(plotmat, 
         scale = "row", 
         show_rownames = TRUE,
         border = FALSE,
         annotation = meta[, c("tumor_percentage_high"), drop = FALSE],
         main = "Top Up/Down-regulated genes in Tumor_percentage_high: high vs low",
         fontsize = 20)

# Create a matrix of normalized expression
sig_up <- resDO_tb_significant %>% arrange(-log2FoldChange) %>% head(50) %>% pull(gene)
sig_down <- resDO_tb_significant %>% arrange(log2FoldChange) %>% head(50) %>% pull(gene)
sig <- c(sig_up, sig_down)

row_annotation <- gene_symbol %>% 
                    as_tibble() %>% 
                    dplyr::filter(gene_id %in% sig)

plotmat <- txi$abundance[c(sig_up, sig_down),] %>% as.data.frame() %>% 
          rownames_to_column(var = "ensembl_gene_id") %>% 
          left_join(gene_symbol, by = c("ensembl_gene_id" = "gene_id")) %>% 
          drop_na(symbol)

plotmat$ensembl_gene_id <- NULL

plotmat <- plotmat %>% column_to_rownames(var = "symbol") %>% as.matrix()

# Color palette
heat.colors <- brewer.pal(6, "YlOrRd")

# Plot heatmap
pheatmap(plotmat, 
         scale = "row", 
         show_rownames = TRUE,
         border = FALSE,
         annotation = meta[, c("response"), drop = FALSE],
         main = "Top 50 Up- and Down- regulated genes in date_of: 20180323 vs 20180228",
         fontsize = 20)

18 Functional analysis

18.1 Biological Process (BP)

bg_genes <- rownames(resResponse)

## Run GO enrichment analysis 
compgo_file <- "data/day8.compgo.up.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
    compGO <- enrichGO(gene = sigResponse_up,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "BP", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
    saveRDS(compGO, compgo_file)
}

dotplot(compGO,
        showCategory = 20, 
        title = "GO (Biological Process) Enrichment \n Analysis for UP in pCR)",
        label_format = 20,
        font.size = 10)

## Output results from GO analysis to a table

print("UP")
## [1] "UP"
results_up <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
nrow(results_up)
## [1] 458
write_csv(results_up, "tables/T20.day8.GO_BP_UP.csv")
compgo_file <- "data/day8.compgo.down.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
    compGO <- enrichGO(gene = sigResponse_down,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "BP", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
    saveRDS(compGO, compgo_file)
}


results_down <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
print("Down")
## [1] "Down"
nrow(results_down)
## [1] 4
write_csv(results_down, "tables/T21.day8.GO_BP_DOWN.csv")
dotplot(compGO,
        showCategory = 20, 
        title = "GO (Biological Process) Enrichment \n Analysis for Down in pCR)",
        label_format = 20,
        font.size = 10)

18.2 Molecular Function (MF)

bg_genes <- rownames(resResponse)

## Run GO enrichment analysis 
compgo_file <- "data/day8.compgo.up.mf.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
    compGO <- enrichGO(gene = sigResponse_up,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "MF", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
    saveRDS(compGO, compgo_file)
}
# image pdf 12 x 12

## Output results from GO analysis to a table
print("UP")
## [1] "UP"
results_up <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
nrow(results_up)
## [1] 28
write_csv(results_up, "tables/T22.day8.GO_MF_UP.csv")

dotplot(compGO,
        showCategory = 20, 
        title = "GO (Molecular Function) Enrichment \n Analysis for UP in pCR)",
        label_format = 20,
        font.size = 10)

compgo_file <- "data/day8.compgo.down.mf.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
  compGO <- enrichGO(gene = sigResponse_down,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "MF", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
   saveRDS(compGO, compgo_file)
}

results_down <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
print("Down")
## [1] "Down"
nrow(results_down)
## [1] 0
write_csv(results_up, "tables/T22.day8.GO_MF_DOWN.csv")

if (nrow(results_down) > 0){
  dotplot(compGO,
        showCategory = 20, 
        title = "GO (Molecular Function) Enrichment \n Analysis for DOWN in pCR)",
        label_format = 20,
        font.size = 10)
}

18.3 Cellular Compartment (CC)

bg_genes <- rownames(resResponse)

## Run GO enrichment analysis 
compgo_file <- "data/day8.compgo.up.cc.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
  compGO <- enrichGO(gene = sigResponse_up,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "CC", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
  saveRDS(compGO, compgo_file)
}

# image pdf 12 x 12

## Output results from GO analysis to a table
print("UP")
## [1] "UP"
results_up <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
nrow(results_up)
## [1] 71
write_csv(results_up, "tables/T23.day8.GO_CC_UP.csv")


dotplot(compGO,
        showCategory = 20, 
        title = "GO (Cellular compartment) Enrichment \n Analysis for UP in pCR)",
        label_format = 20,
        font.size = 10)

compgo_file <- "data/day8.compgo.down.cc.RDS"
if (file.exists(compgo_file)){
    compGO <- readRDS(compgo_file)
}else{
    compGO <- enrichGO(gene = sigResponse_down,
                   universe = bg_genes,
                   keyType = "ENSEMBL",
                   OrgDb = "org.Hs.eg.db", 
                   ont = "CC", 
                   qvalueCutoff  = 0.05, 
                   pAdjustMethod = "BH",
                   readable = TRUE)
    saveRDS(compGO, compgo_file)
}

results_down <- data.frame(compGO@result) %>% dplyr::filter(p.adjust < 0.05)
print("Down")
## [1] "Down"
nrow(results_down)
## [1] 0
write_csv(results_up, "tables/T24.day8.GO_CC_DOWN.csv")

if (nrow(results_down) > 0){
dotplot(compGO,
        showCategory = 20, 
        title = "GO (Cellular compartment) Enrichment \n Analysis for DOWN in pCR)",
        label_format = 20,
        font.size = 10)
}

19 R session

sessionInfo()
## R version 4.0.5 (2021-03-31)
## Platform: x86_64-redhat-linux-gnu (64-bit)
## Running under: Fedora 32 (Workstation Edition)
## 
## Matrix products: default
## BLAS/LAPACK: /usr/lib64/libopenblas-r0.3.12.so
## 
## locale:
##  [1] LC_CTYPE=en_CA.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_CA.UTF-8        LC_COLLATE=en_CA.UTF-8    
##  [5] LC_MONETARY=en_CA.UTF-8    LC_MESSAGES=en_CA.UTF-8   
##  [7] LC_PAPER=en_CA.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_CA.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] parallel  stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] writexl_1.4.0               clusterProfiler_3.18.1     
##  [3] org.Hs.eg.db_3.12.0         ensembldb_2.14.1           
##  [5] AnnotationFilter_1.14.0     GenomicFeatures_1.42.3     
##  [7] AnnotationDbi_1.52.0        AnnotationHub_2.22.1       
##  [9] BiocFileCache_1.14.0        dbplyr_2.1.1               
## [11] knitr_1.33                  ggrepel_0.9.1              
## [13] tximport_1.18.0             DEGreport_1.26.0           
## [15] pheatmap_1.0.12             RColorBrewer_1.1-2         
## [17] forcats_0.5.1               stringr_1.4.0              
## [19] dplyr_1.0.7                 purrr_0.3.4                
## [21] readr_1.4.0                 tidyr_1.1.3                
## [23] tibble_3.1.2                ggplot2_3.3.5              
## [25] tidyverse_1.3.1             DESeq2_1.30.1              
## [27] SummarizedExperiment_1.20.0 Biobase_2.50.0             
## [29] MatrixGenerics_1.2.1        matrixStats_0.59.0         
## [31] GenomicRanges_1.42.0        GenomeInfoDb_1.26.7        
## [33] IRanges_2.24.1              S4Vectors_0.28.1           
## [35] BiocGenerics_0.36.1        
## 
## loaded via a namespace (and not attached):
##   [1] utf8_1.2.1                    tidyselect_1.1.1             
##   [3] RSQLite_2.2.7                 grid_4.0.5                   
##   [5] BiocParallel_1.24.1           scatterpie_0.1.6             
##   [7] munsell_0.5.0                 withr_2.4.2                  
##   [9] colorspace_2.0-2              GOSemSim_2.16.1              
##  [11] highr_0.9                     rstudioapi_0.13              
##  [13] DOSE_3.16.0                   labeling_0.4.2               
##  [15] lasso2_1.2-21.1               GenomeInfoDbData_1.2.4       
##  [17] polyclip_1.10-0               mnormt_2.0.2                 
##  [19] farver_2.1.0                  bit64_4.0.5                  
##  [21] downloader_0.4                vctrs_0.3.8                  
##  [23] generics_0.1.0                xfun_0.22                    
##  [25] R6_2.5.0                      graphlayouts_0.7.1           
##  [27] clue_0.3-59                   locfit_1.5-9.4               
##  [29] bitops_1.0-7                  cachem_1.0.5                 
##  [31] reshape_0.8.8                 fgsea_1.16.0                 
##  [33] DelayedArray_0.16.3           assertthat_0.2.1             
##  [35] promises_1.2.0.1              scales_1.1.1                 
##  [37] ggraph_2.0.5                  enrichplot_1.10.2            
##  [39] gtable_0.3.0                  Cairo_1.5-12.2               
##  [41] conquer_1.0.2                 tidygraph_1.2.0              
##  [43] MatrixModels_0.5-0            rlang_0.4.11                 
##  [45] genefilter_1.72.1             GlobalOptions_0.1.2          
##  [47] splines_4.0.5                 rtracklayer_1.50.0           
##  [49] lazyeval_0.2.2                broom_0.7.8                  
##  [51] BiocManager_1.30.16           yaml_2.2.1                   
##  [53] reshape2_1.4.4                modelr_0.1.8                 
##  [55] backports_1.2.1               httpuv_1.6.1                 
##  [57] qvalue_2.22.0                 tools_4.0.5                  
##  [59] psych_2.1.6                   logging_0.10-108             
##  [61] ellipsis_0.3.2                ggdendro_0.1.22              
##  [63] Rcpp_1.0.7                    plyr_1.8.6                   
##  [65] progress_1.2.2                zlibbioc_1.36.0              
##  [67] RCurl_1.98-1.3                prettyunits_1.1.1            
##  [69] openssl_1.4.4                 viridis_0.6.1                
##  [71] GetoptLong_1.0.5              cowplot_1.1.1                
##  [73] haven_2.4.1                   cluster_2.1.1                
##  [75] fs_1.5.0                      magrittr_2.0.1               
##  [77] magick_2.7.2                  data.table_1.14.0            
##  [79] DO.db_2.9                     SparseM_1.81                 
##  [81] circlize_0.4.13               reprex_2.0.0                 
##  [83] tmvnsim_1.0-2                 ProtGenerics_1.22.0          
##  [85] hms_1.1.0                     mime_0.11                    
##  [87] evaluate_0.14                 xtable_1.8-4                 
##  [89] XML_3.99-0.6                  readxl_1.3.1                 
##  [91] gridExtra_2.3                 shape_1.4.6                  
##  [93] compiler_4.0.5                biomaRt_2.46.3               
##  [95] shadowtext_0.0.8              crayon_1.4.1                 
##  [97] htmltools_0.5.1.1             later_1.2.0                  
##  [99] geneplotter_1.68.0            lubridate_1.7.10             
## [101] DBI_1.1.1                     tweenr_1.0.2                 
## [103] ComplexHeatmap_2.6.2          MASS_7.3-53.1                
## [105] rappdirs_0.3.3                Matrix_1.3-4                 
## [107] cli_3.0.1                     igraph_1.2.6                 
## [109] pkgconfig_2.0.3               rvcheck_0.1.8                
## [111] GenomicAlignments_1.26.0      xml2_1.3.2                   
## [113] annotate_1.68.0               XVector_0.30.0               
## [115] rvest_1.0.0                   digest_0.6.27                
## [117] ConsensusClusterPlus_1.54.0   Biostrings_2.58.0            
## [119] rmarkdown_2.6                 cellranger_1.1.0             
## [121] fastmatch_1.1-0               edgeR_3.32.1                 
## [123] curl_4.3.2                    quantreg_5.86                
## [125] shiny_1.6.0                   Rsamtools_2.6.0              
## [127] rjson_0.2.20                  lifecycle_1.0.0              
## [129] nlme_3.1-152                  jsonlite_1.7.2               
## [131] viridisLite_0.4.0             askpass_1.1                  
## [133] limma_3.46.0                  fansi_0.5.0                  
## [135] pillar_1.6.1                  lattice_0.20-41              
## [137] Nozzle.R1_1.1-1               fastmap_1.1.0                
## [139] httr_1.4.2                    survival_3.2-10              
## [141] GO.db_3.12.1                  interactiveDisplayBase_1.28.0
## [143] glue_1.4.2                    png_0.1-7                    
## [145] BiocVersion_3.12.0            bit_4.0.4                    
## [147] ggforce_0.3.3                 stringi_1.7.3                
## [149] blob_1.2.1                    memoise_2.0.0